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Biomedical subjects

W D Reed

Publications and source records attributed to W D Reed.

At least 73 records · Page 4Linked to original sources
PubMed1977

Immune responses to the hepatitis B surface antigen and liver-specific lipoprotein in acute type B hepatitis.

A serial prospective study of cellular immunity to HBsAg and liver-specific membrane lipoprotein was undertaken in 21 adults with acute hepatitis type B. Cellular immunity to HBsAg as determined by leucocyte migration inhibition with partially purified HBsAg as antigen was detected in all the patients during the recovery phase of the illness and was already detectable at the time of admission in 13 (62%) of the cases. In five of the remaining eight the titre of HBsAg in the serum at this time was high and in the whole series there was an inverse correlation between the degree of migration inhibition on admission and the peak HBsAg titre suggesting that antigen or possibly antigen/antibody complexes might be interfering with the demonstration of cellular immunity in vitro. Using a combination of minimum migration index recorded during the recovery period peak HBsAg titre, it was possible to compute the peak aspartate aminotransferase level with reasonable accuracy, a finding consistent with the hypothesis that the severity of the illness is related to both the number of infected hepatocytes and the vigour of the immune response to HBsAg. Evidence of an immune response to the liver-specific hepatocyte membrane lipoprotein was present in 50% of the patients tested at the time of admission, but was transient, having disappeared in every case by four weeks. The minimum migration index recorded with HBsAg as antigen was significantly lower in those with detectable sensitisation to the lipoprotein and it is possible that this autoimmune reaction is also generated by the interaction of T cells with viral antigenic determinants on the liver cell surface.

Acute Disease↗
PubMed1976

Ability of exogenous alanine to lower blood beta-hydroxybutyrate during development in rats.

The intraperitoneal injection of L-alanine into 10- and 21-day-old fed Wistar rats resulted in blood alanine levels of 3-5 mM and in a rapid, significant decrease in the concentration of beta-hydroxybutyrate (betaOHB) in blood. The drop in betaOHB was unrelated to changes in the concentrations of pyruvate, lactate, or glucose. The intraperitoneal injection of glutamate at the same age had no effect on betaOHB values. In 2-day-old pups and in older weaned animals, the injection of alanine had either no effect or increased the levels of betaOHB in blood. The most rapid disappearance of exogenous alanine occurred in 35-day-old and not in 10- and 21-day-old rats. These data suggest that alanine has a depressant effect on the level of circulating betaOHB at a specific stage in the animal's maturation.

Alanine↗
PubMed1975

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.

Acetylation↗
PubMed1975

Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis.

Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase.

Adenosine Monophosphate↗
PubMed1975

Molecular and catalytic properties of mitochondrial (ketogenic) 3-hydroxy-3-methylglutaryl coenzyme A synthase of liver.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase has been purified to homogeneity from avian liver. The enzyme in dilute phosphate buffer, pH 7.0, has an S20,w of 5.7 S and a molecular weight of 105,000 determined by sedimentation equilibrium; the presence of 0.1 M KCl causes dissociation to a form one-half that size, i.e. about 57,000 daltons. Since the subunit molecular weight of the synthase determined by the dodecyl sulfate acrylamide gel method is 53,000, it appears that the native enzyme is a dimer composed of weight-homogeneous subunits. A number of molecular and catalytic properties allow the mitochondrial and cytoplasmic 3-hydroxy-3-methylglutaryl-CoA synthases to be distinguished. The pI of the homogeneous mitochondrial enzyme is 7.2. This value, while identical to that of the single isoelectric-focusing species of broken mitochondrial preparations, differs from those of the multiple 3-hydroxy-3-methylglutaryl-CoA synthases found in the cytoplasmic fraction which exhibit pI values of 4.8 and 6.7. Rabbit antibodies against the purified mitochondrial synthase are capable of precipitating the mitochondrial, but not the cytoplasmic, synthase(s) of avian liver. Finally, the synthase differ kinetically in their responses to divalent magnesium ion, the mitochondrial enzyme being inhibited and the cytoplasmic enzyme(s) activated. It is proposed that the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase of liver functions in ketogenesis while its cytoplasmic counterpart participates in cholesterogenesis (Clinken-Beard, K. D., Sugiyama, T., Reed, W. D. and Lane, M.D. (1975) J. Biol. Chem. 250, 3124-3134).

Animals↗
PubMed1975

Cellular and humoral immunity to hepatitis-B surface antigen in active chronic hepatitis.

The hepatitis-B surface antigen (HBsAG) may be persistently present in the serum in a few cases of active chronic hepatitis but the cause of the disease in most patients is unknown. In a study of 39 HBsAg-negative cases cell-mediated immunity to HBsAg was observed in 24 (62%), suggesting a high frequency of previous infection with the hepatitis-B virus. Hepatitis-B surface antibody was detectable by radioimmunoassay in six patients, in all of whom complexes of HBsAg were present in the serum on electron microscopy. Out of 12 patients with HBsAg-positive active chronic hepatitis who were also studied eight, including all those untreated at the time, showed a cellular response to the antigen. Evidence of sensitization to a liver-specific cell surface lipoprotein was found with similar frequency in the two groups. These results are consistent with the hypothesis that hepatitis-B virus infection is important in initiating the disease in many cases of active chronic hepatitis and that sensitization to the liver cell membrane antigen is the autoimmune process responsible for the perpetuation of the liver injury.

Antibodies, Viral↗
PubMed1975

Cell-mediated immunity to hepatitis B surface antigen in blood donors with persistent antigenaemia.

Cellular immunity to the hepatitis B surface antigen (HBsAg) and a liver-specific lipoprotein was studied, using the leucocyte migration test, in 38 asymptomatic blood donors found to have HBsAg in the serum. Sensitization to HBsAg was found in 26% and was related to the presence of liver damage, being detected in 47% of those with elevated serum aspartate aminotransferase but in only 13% with normal enzyme levels. The frequency of sensitization to this antigen in those with chronic persistent or chronic aggressive hepatitis on biopsy was also higher than in those with unrelated or minimal changes. The findings using the liver-specific lipoprotein as antigen were similar and there was a correlation between the results obtained with this and the hapatitis B surface antigen. This study supports the hypothesis that a T-lymphocyte response to hepatitis B virus antigen can initiate an autoimmune reaction to antigens such as liver-specific lipoprotein on the hepatocyte surface, and that this reaction may be of importance in the production of chronic liver damage. In the absence of the T-cell response, the autoimmune reaction cannot occur and the virus is able to establish a harmless symbiotic union with the host.

Aspartate Aminotransferases↗
PubMed1974

Regulation of muscle phosphorylase b kinase activity by inorganic phosphate and calcium ions.

The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P(i) and Ca(2+) was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P(i) increased activity severalfold. The effect was concentration-dependent, with an apparent K(m) of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg(2+) in addition to P(i). The apparent K(m) for Mg(2+) was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P(i), and was strongly inhibited by K(+), Na(+) and Li(+). At physiological concentrations, Ca(2+) activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca(2+) of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca(2+), and showed a large increase in activity over a narrow range of Ca(2+) concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca(2+), and had a smaller increase over a wide range of Ca(2+) concentration. The concentrations of Ca(2+) required for half-activation were 0.1 and 1mum for the blowfly and rabbit enzymes respectively. The pH-activity profiles of the non-activated, phosphate- and Ca(2+)-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.

Adenosine Triphosphate↗
PubMed1973

Cell-mediated immune response in primary biliary cirrhosis to a protein fraction from human bile.

Cell-mediated immune responses to a protein fraction of human bile have been demonstrated, using the leucocyte migration test, in eight out of 10 patients with primary biliary cirrhosis but in only three out of nine with active chronic hepatitis. In the latter condition sensitization to a liver-specific hepatocellular antigen was found more frequently (five out of nine patients) than in primary biliary cirrhosis (two out of 10). These results, as well as the granuloma formation observed histologically, suggest that the initial bile duct lesion in primary biliary cirrhosis may be associated with a cell-mediated response to antigens-perhaps derived from bile duct epithelial cells-which may be normal constituents of hepatic bile.

Antigens↗